The ADP-ribosylation factors (ARFs) are 20 kDa guanine nucleotide-binding proteins that were first identified based on their ability to enhance the cholera toxin-catalyzed ADP-ribosylation of Gs-alpha. ARFs isolated from various mammalian tissues had different phospholipid/detergent and nucleotide requirements for activity and stability. Immunological studies using anti-ARF polyclonal antibodies identified membrane and soluble proteins present in all mammalian tissues; the relative amounts of these immunoreactive proteins varied in a tissue-dependent fashion. The fact that six mammalian ARF-like proteins, five human and one bovine, have been identified based on their cDNA and deduced amino acid sequences suggested that different ARFs predominate in different tissues. To investigate whether individual ARFs differ in their requirements for activity, we examined the effects of phospholipids/detergents and GTP concentration on the stimulation of cholera toxin by both native and recombinant ARFs. Although recombinant ARFs expressed in E. coli enhanced the ADP- ribosyltransferase activity of cholera toxin, the requirements for maximal ARF activity and GTP binding were different for the two ARFs. Recombinant ARFs 2 and 6 differed in the concentrations of GTP required for half-maximal activity, the time course for toxin stimulation, and their requirements for phospholipid/detergent. In summary,-these data are consistent with our belief that the relative of specific ARFs differ in different cells and that individual ARFs may have different functions.